Although the major factors affecting renal Ca handling and their tubular sites of action have been well studied, available information regarding cellular mechanism(s) of their effects is limited. Little is known related to cytosolic Ca movement from lumen to peritubular space or vice versa. The recently available Ca-sensitive fluorescent dyes permitting estimates of Ca2+ in living cells provide an excellent opportunity to examine Ca transport-related processes in response to administration of hormones, pharmacologic agents or manipulations of medium ionic composition. The in-vitro microperfusion technology permits separate, selective and complete control of the chemical composition of luminal and peritubular fluids, enabling one to define not only the effects but also the membrane site of action. By virtue of its high Ca-, Mg-ATPase activity, the abundance of vitamin D-dependent Ca-binding protein and its response to stimulatory effects of PTH on Ca absorption, the rabbit connecting tubules (CNT) is perhaps the single most critical segment regulating renal Ca excretion and systemic Ca homeostasis. Measurements of lumen-to-bath and bath-to-lumen 45Ca fluxes, simultaneous with Ca2+ by epifluorescence miroscopy in microperfused fura 2-loaded CNT, will be done:- (1) To define the mechanism for PTH-stimulated increase in Ca2+. 2) To examine the effects of alterations in apical membrane Na entry on lumen-to-bath Ca flux and on Ca2+ in the absence of altered luminal voltage. (3) To evaluate the separate role of luminal and peritubular PO4 on basal and PTH-stimulated Ca2+ and on Ca transport. (4) To test the hypothesis that changes in transepithelial Ca transport produced by luminal PO4 and HCO3 are mediated by alterations in the H+ buffers. (5) To evaluate the effects and mechanism of action of 1,25(OH)2D3 on Ca2+ and on bi-directional Ca fluxes. These experiments will test the overall hypothesis that in the CNT, Ca2+ is primarily determined by apical Ca entry, as modulated by humoral and ionic factors. The Ca2+, in turn, regulates Ca exit across the BLM and therefore determines the overall rate of transcellular Ca absorption. The long-term goal is to study the cellular mechanism(s) controlling Ca transport. These results will better define the pathophysiology in various conditions characterized by abnormal rates of Ca excretion or deranged mineral metabolism and will improve our therapeutic modalities.